1. Technical Field
The present invention relates generally to compositions and methods useful for treating conditions associated with defects in cell proliferation, cell differentiation, and cell survival. The invention is more particularly related to double-stranded RNA polynucleotides that interfere with expression of protein tyrosine phosphatases, and polypeptide variants thereof. The invention is also particularly related to double-stranded RNA polynucleotides that interfere with expression of MAP kinases and MAP kinase kinases and chemotherapeutic target polypeptides, and polypeptide variants thereof. The present invention is also related to the use of such RNA polynucleotides to alter activation of signal transduction pathway components or to alter cellular metabolic processes that lead to proliferative responses, cell differentiation and development, and cell survival.
2. Description of the Related Art
Reversible protein tyrosine phosphorylation, coordinated by the action of protein tyrosine kinases (PTKs) that phosphorylate certain tyrosine residues in polypeptides, and protein tyrosine phosphatases (PTPs) that dephosphorylate certain phosphotyrosine residues, is a key mechanism in regulating many cellular activities. It is becoming apparent that the diversity and complexity of the PTPs and PTKs are comparable, and that PTPs are equally important in delivering both positive and negative signals for proper function of cellular machinery. Regulated tyrosine phosphorylation contributes to specific pathways for biological signal transduction, including those associated with cell division, cell survival, apoptosis, proliferation and differentiation. Defects and/or malfunctions in these pathways may underlie certain disease conditions for which effective means for intervention remain elusive, including for example, malignancy, autoimmune disorders, diabetes, obesity and infection.
The protein tyrosine phosphatase (PTP) family of enzymes consists of more than 100 structurally diverse proteins in vertebrates, including almost 40 human PTPs that have in common the conserved 250 amino acid PTP catalytic domain, but which display considerable variation in their non-catalytic segments (Charbonneau and Tonks, 1992 Annu. Rev. Cell Biol. 8:463-493; Tonks, 1993 Semin. Cell Biol. 4:373-453; Andersen et al., Mol. Cell Biol. 21:7117-36 (2001)). This structural diversity presumably reflects the diversity of physiological roles of individual PTP family members, which in certain cases have been demonstrated to have specific functions in growth, development and differentiation (Desai et al., 1996 Cell 84:599-609; Kishihara et al., 1993 Cell 74:143-156; Perkins et al., 1992 Cell 70:225-236; Pingel and Thomas, 1989 Cell 58:1055-1065; Schultz et al., 1993 Cell 73:1445-1454). The PTP family includes receptor-like and non-transmembrane enzymes that exhibit exquisite substrate specificity in vivo and that are involved in regulating a wide variety of cellular signaling pathways (Andersen et al., Mol. Cell. Biol. 21:7117 (2001); Tonks and Neel, Curr. Opin. Cell Biol. 13:182 (2001)). PTPs thus participate in a variety of physiologic functions, providing a number of opportunities for therapeutic intervention in physiologic processes through alteration (i.e., a statistically significant increase or decrease) or modulation (e.g., up-regulation or down-regulation) of PTP activity.
Although recent studies have also generated considerable information regarding the structure, expression and regulation of PTPs, the nature of many tyrosine phosphorylated substrates through which the PTPs exert their effects remains to be determined. Studies with a limited number of synthetic phosphopeptide substrates have demonstrated some differences in the substrate selectivities of different PTPs (Cho et al., 1993 Protein Sci. 2: 977-984; Dechert et al., 1995 Eur. J. Biochem. 231:673-681). Analyses of PTP-mediated dephosphorylation of PTP substrates suggest that catalytic activity may be favored by the presence of certain amino acid residues at specific positions in the substrate polypeptide relative to the phosphorylated tyrosine residue (Salmeen et al., 2000 Molecular Cell 6:1401; Myers et al., 2001 J. Biol. Chem. 276:47771; Myers et al., 1997 Proc. Natl. Acad. Sci. USA 94:9052; Ruzzene et al., 1993 Eur. J. Biochem. 211:289295; Zhang et al., 1994 Biochemistry 33:2285-2290). Thus, although the physiological relevance of the substrates used in these studies is unclear, PTPs display a certain level of substrate selectivity in vitro.
The PTP family of enzymes contains a common evolutionarily conserved segment of approximately 250 amino acids known as the PTP catalytic domain. Within this conserved domain is a unique signature sequence motif, CX5R (SEQ ID NO:1), that is invariant among all PTPs. In a majority of PTPs, an 11 amino acid conserved sequence ([IIV]HCXAGXXR[S/T)G (SEQ ID NO: 2)) containing the signature sequence motif is found. The cysteine residue in this motif is invariant in members of the family and is essential for catalysis of the phosphotyrosine dephosphorylation reaction. It functions as a nucleophile to attack the phosphate moiety present on a phosphotyrosine residue of the incoming substrate. If the cysteine residue is altered by site-directed mutagenesis to serine (e.g., in cysteine-to-serine or “CS” mutants) or alanine (e.g., cysteine-to-alanine or “CA” mutants), the resulting PTP is catalytically deficient but retains the ability to complex with, or bind, its substrate, at least in vitro.
The CS mutant of one PTP, PTP1B (PTP-1B), is an example of such a PTP. Catalytically deficient mutants of such enzymes that are capable of forming stable complexes with phosphotyrosyl polypeptide substrates may be derived by mutating a wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue and replacing it with an amino acid that does not cause significant alteration of the Km of the enzyme but that results in a reduction in Kcat, as disclosed, for example, in U.S. Pat. Nos. 5,912,138 and 5,951,979, in U.S. application Ser. No. 09/323,426 and in PCT/US97/13016 and PCT/JUS00/14211. For instance, mutation of Asp 181 in PTP1B to alanine to create the aspartate-to-alanine (D to A or DA) mutant PTP1B-D181A results in a PTP1B “substrate trapping” mutant enzyme that forms a stable complex with its phosphotyrosyl polypeptide substrate (e.g., Flint et al., 1997 Proc. Natl. Acad. Sci. 94:1680). Substrates of other PTPs can be identified using a similar substrate trapping approach, for example substrates of the PTP family members PTP-PEST (Garton et al., 1996 J. Mol. Cell. Biol. 16:6408), TCPTP (Tiganis et al., 1998 Mol. Cell Biol. 18:1622), PTP-HSCF (Spencer et al., 1997 J. Cell Biol. 138:845), and PTP-H1 (Zhang et al., 1999 J. Biol. Chem. 274:17806).
Mitogen-activated protein kinases (MAP-kinases) are components of conserved cellular signal transduction pathways that have a variety of conserved members and that that are integral to the cell's response to stimuli such as growth factors, hormones, cytokines, and environmental stresses. MAP-kinases are activated by phosphorylation by MAP-kinase kinases at a dual phosphorylation motif that has the sequence Thr-X-Tyr, in which phosphorylation at the tyrosine and threonine residues is required for activity. Activated MAP-kinases phosphorylate several transduction targets, including effector protein kinases and transcription factors. Inactivation of MAP-kinases is mediated by dephosphorylation at the Thr-X-Tyr site by dual-specificity phosphatases referred to as MAP-kinase phosphatases. In higher eukaryotes, the physiological role of MAP-kinase signaling has been correlated with cellular events such as proliferation, oncogenesis, development, and differentiation. Accordingly, the ability to regulate signal transduction via these pathways could lead to the development of treatments and preventive therapies for human diseases associated with MAP-kinase signaling, such as cancer.
Dual-specificity protein tyrosine phosphatases (dual-specificity phosphatases) dephosphorylate both phosphotyrosine and phosphothreonine/serine residues (Walton et al., Ann. Rev. Biochem. 62:101-120, 1993). More than 50 dual-specificity phosphatases that dephosphorylate and inactivate a MAP-kinase have been identified (Shen et al., Proc. Natl. Acad. Sci. USA 98:13613-18 (2001)), including MKP-1 (WO 97/00315; Keyse and Emslie, Nature 59:644-647 (1992)); MKP-2 (WO97/00315); MKP-4, MKP-5, MKP-7, Hb5 (WO 97/06245); PAC1 (Ward et al., Nature 367:651-654 (1994)); HVH2 (Guan and Butch, J. Biol. Chem. 270:7197-7203 (1995)); and PYST1 (Groom et al., EMBO J. 15:3621-3632 (1996)). These dual-specificity phosphatases differ in expression, tissue and subcellular distribution, and specificity for MAP-kinase family members. Expression of certain dual-specificity phosphatases is induced by stress or mitogens, but others appear to be expressed constitutively in specific cell types. The regulation of dual-specificity phosphatase expression and activity is critical for control of MAP-kinase mediated cellular functions, including cell proliferation, cell differentiation and cell survival. For example, dual-specificity phosphatases may function as negative regulators of cell proliferation. It is likely that there are many such dual-specificity phosphatases, with varying specificity with regard to cell type or activation.
In contrast to the role of most dual-specificity phosphatases to inactivate MAP-kinases, one enzyme, herein referred to as dual-specificity phosphatase 3 (DSP-3), has been reported to have the capability to function as a selective activator of the JNK MAP-kinase signaling pathway (Shen et al., supra; WO 01/21812). DSP-3 appears also to affect the activity of other kinases involved in the JNK pathway (Shen et al., supra; WO 01/21812). For example, overexpression of DSP-3 leads to activation of MKK4, a MAP-kinase kinase that functions upstream of JNK (Shen et al., supra; Lawler et al., Curr. Biol. 8:1387-90 (1998); Yang et al., Proc. Natl. Acad. Sci. USA 94: 3004-3009 (1997)).
Activation of JNK is believed to be involved in several physiological processes, including embryonic morphogenesis, cell survival, and apoptosis. A number of JNK signaling pathway substrates have been identified, including c-Jun, ATF2, ELK-1 and others. JNK signaling has also been associated with various disease conditions, such as tumor development, ischemia and reperfusion injury, diabetes, hyperglycemia-induced apoptosis, cardiac hypertrophy, inflammation, and neurodegenerative disorders.
One non-transmembrane PTP, PTP1B, recognizes several tyrosine-phosphorylated proteins as substrates, many of which are involved in human disease. For example, therapeutic inhibition of PTP1B in the insulin signaling pathway may serve to augment insulin action, thereby ameliorating the state of insulin resistance common in Type II diabetes patients. PTP1B acts as a negative regulator of signaling that is initiated by several growth factor/hormone receptor PTKs, including p210 Bcr-Abl (LaMontagne et al., Mol. Cell Biol. 18:2965-75 (1998); LaMontagne et al., Proc. Natl. Acad. Sci. USA 95:14094-99 (1998)), receptor tyrosine kinases, such as EGF receptor, PDGF receptor, and insulin receptor (IR) (Tonks et al., Curr. Opin. Cell Biol. 13:182-95 (2001)), and JAK family members such as Jak2 and others (Myers et al., J. Biol. Chem. 276:47771-74 (2001)), as well as signaling events induced by cytokines (Tonks and Neel, 2001). Activity of PTP1B is regulated by modifications of several amino acid residues, such as phosphorylation of Ser residues (Brautigan and Pinault, 1993; Dadke et al., 2001; Flint et al., 1993), and oxidation of the active Cys residue in its catalytic motif (Lee et al., 1998; Meng et al., 2002) which is evolutionary conserved among protein tyrosine phosphatases and dual phosphatase family members (Andersen et al., 2001).
Disruption of the murine PTP1B gene homolog in a knock-out mouse model results in PTP1B−/− mice exhibiting enhanced insulin sensitivity, decreased levels of circulating insulin and glucose, and resistance to weight gain even on a high-fat diet, relative to control animals having at least one functional PTP1B gene (Elchebly et al., Science 283:1544 (1999)). Insulin receptor hyperphosphorylation has also been detected in certain tissues of PTP1B deficient mice, consistent with a PTP1B contribution to the physiologic regulation of insulin and glucose metabolism (Id.). PTP-1B-deficient mice exhibit decreased adiposity (reduced fat cell mass but not fat cell number), increased basal metabolic rate and energy expenditure, and enhanced insulin-stimulated glucose utilization (Klaman et al., 2000 Mol. Cell. Biol. 20:5479). Additionally, altered PTP activity has been correlated with impaired glucose metabolism in other biological systems (e.g., McGuire et al., Diabetes 40:939 (1991); Myerovitch et al., J. Clin. Invest. 84:976 (1989); Sredy et al., Metabolism 44:1074 (1995)), including PTP involvement in biological signal transduction via the insulin receptor (see, e.g., WO 99/46268 and references cited therein).
An integration of crystallographic, kinetic, and PTP1B-peptide binding assays illustrated the interaction of PTP1B and insulin receptor (IR) (Salmeen et al., Mol. Cell 6:1401-12 (2000)). The insulin receptor (IR) comprises two extracellular α subunits and two transmembrane β subunits. Activation of the receptor results in autophosphorylation of tyrosine residues in both β subunits, each of which contains a protein kinase domain. Extensive interactions that form between PTP1B and insulin receptor kinase (IRK) encompass tandem pTyr residues at 1162 and 1163 of IRK, such that pTyr-1162 is located in the active site of PTP1B (id.). The Asp/Glu-pTyr-pTyr-Arg/Lys motif has been implicated for optimal recognition by PTP1B for IRK. This motif is also present in other receptor PTKs, including Trk, FGFR, and Axl. In addition, this motif is found in the JAK family of PTKs, members of which transmit signals from cytokine receptors, including a classic cytokine receptor that is recognized by the satiety hormone leptin (Touw et al., Mol. Cell. Endocrinol. 160:1-9 (2000)).
Changes in the expression levels of PTP1B have been observed in several human diseases, particularly in diseases associated with disruption of the normal patterns of tyrosine phosphorylation. For example, the expression of PTP1B is induced specifically by the p210 Bcr-Abl oncoprotein, a PTK that is directly responsible for the initial manifestations of chronic myelogenous leukemia (CML) (LaMontagne et al., Mol. Cell. Biol. 18:2965-75 (1998); LaMontagne et al., Proc. Natl. Acad. Sci. USA 95:14094-99 (1998)). Expression of PTPB1 in response to this oncoprotein is regulated, in part, by transcription factors Sp1, Sp3, and Egr-1 (Fukada et al., J. Biol. Chem. 276:25512-19 (2001)). These transcription factors have been shown to bind to a p210 Bcr-Abl responsive sequence (PRS) in the human PTP1B promoter, located between 49 to −37 base pairs from the transcription start site, but do not appear to mediate certain additional, independent PTP1B transcriptional events, for which neither transcription factor(s) nor transcription factor recognition element(s) have been defined (id.).
Diabetes mellitus is a common, degenerative disease affecting 5-10% of the human population in developed countries, and in many countries, it may be one of the five leading causes of death. Approximately 2% of the world's population has diabetes, the overwhelming majority of cases (>97%) being type 2 diabetes and the remainder being type 1. In type 1 diabetes, which is frequently diagnosed in children or young adults, insulin production by pancreatic islet beta cells is destroyed. Type 2 diabetes, or “late onset” or “adult onset” diabetes, is a complex metabolic disorder in which cells and tissues cannot effectively use available insulin; in some cases insulin production is also inadequate. At the cellular level, the degenerative phenotype that may be characteristic of late onset diabetes mellitus includes, for example, impaired insulin secretion and decreased insulin sensitivity, i.e., an impaired response to insulin.
Studies have shown that diabetes mellitus may be preceded by or is associated with certain related disorders. For example, an estimated forty million individuals in the U.S. suffer from late onset impaired glucose tolerance (IGT). IGT patients fail to respond to glucose with increased insulin secretion. Each year a small percentage (5-10%) of IGT individuals progress to insulin deficient non-insulin dependent diabetes (NIDDM). Some of these individuals further progress to insulin dependent diabetes mellitus (IDDM). NIDDM and IDDM are associated with decreased release of insulin by pancreatic beta cells and/or a decreased response to insulin by cells and tissues that normally exhibit insulin sensitivity. Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include obesity, vascular pathologies, and various neuropathies, including blindness and deafness.
Type 1 diabetes is treated with lifelong insulin therapy, which is often associated with undesirable side effects such as weight gain and an increased risk of hypoglycemia. Current therapies for type 2 diabetes (NIDDM) include altered diet, exercise therapy, and pharmacological intervention with injected insulin or oral agents that are designed to lower blood glucose levels. Examples of such presently available oral agents include sulfonylureas, biguanides, thiazolidinediones, repaglinide, and acarbose, each of which alters insulin and/or glucose levels. None of the current pharmacological therapies, however, controls the disease over its full course, nor do any of the current therapies correct all of the physiological abnormalities in type 2 NIDDM, such as impaired insulin secretion, insulin resistance, and excessive hepatic glucose output. In addition, treatment failures are common with these agents, such that multi-drug therapy is frequently necessary.
In certain metabolic diseases or disorders, one or more biochemical processes, which may be either anabolic or catabolic (e.g., build-up or breakdown of substances, respectively), are altered (e.g., increased or decreased in a statistically significant manner) or modulated (e.g., up- or down-regulated to a statistically significant degree) relative to the levels at which they occur in a disease-free or normal subject such as an appropriate control individual. The alteration may result from an increase or decrease in a substrate, enzyme, cofactor, or any other component in any biochemical reaction involved in a particular process. Altered (i.e., increased or decreased in a statistically significant manner relative to a normal state) PTP activity can underlie certain disorders and suggests a PTP role in certain metabolic diseases.
RNA interference (RNAi) is a polynucleotide sequence-specific, post-transcriptional gene silencing mechanism effected by double-stranded RNA that results in degradation of a specific messenger RNA (mRNA), thereby reducing the expression of a desired target polypeptide encoded by the mRNA (see, e.g., WO 99/32619; WO 01/75164; U.S. Pat. No. 6,506,559; Fire et al., Nature 391:806-11 (1998); Sharp, Genes Dev. 13:139-41 (1999); Elbashir et al. Nature 411:494-98 (2001); Harborth et al., J. Cell Sci. 114:4557-65 (2001)). RNAi is mediated by double-stranded polynucleotides as also described hereinbelow, for example, double-stranded RNA (dsRNA), having sequences that correspond to exonic sequences encoding portions of the polypeptides for which expression is compromised. RNAi reportedly is not effected by double-stranded RNA polynucleotides that share sequence identity with intronic or promoter sequences (Elbashir et al., 2001). RNAi pathways have been best characterized in Drosophila and Caenorhabditis elegans, but “small interfering RNA” (siRNA) polynucleotides that interfere with expression of specific polypeptides in higher eukaryotes such as mammals (including humans) have also been considered (e.g., Tuschl, 2001 Chembiochem. 2:239-245; Sharp, 2001 Genes Dev. 15:485; Bernstein et al., 2001 RNA 7:1509; Zamore, 2002 Science 296:1265; Plasterk, 2002 Science 296:1263; Zamore 2001 Nat. Struct. Biol. 8:746; Matzke et al., 2001 Science 293:1080; Scadden et al., 2001 EMBO Rep. 2:1107).
According to a current non-limiting model, the RNAi pathway is initiated by ATP-dependent, processive cleavage of long dsRNA into double-stranded fragments of about 18-27 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, etc.) nucleotide base pairs in length, called small interfering RNAs (siRNAs) (see review by Hutvagner et al., Curr. Opin. Gen. Dev. 12:225-32 (2002); Elbashir et al., 2001; Nykänen et al., Cell 107:309-21 (2001); Zamore et al., Cell 101:25-33 (2000); Bass, Cell 101:235-38 (2000)). In Drosophila, an enzyme known as “Dicer” cleaves the longer double-stranded RNA into siRNAs; Dicer belongs to the RNase III family of dsRNA-specific endonucleases (WO 01/68836; Bernstein et al., Nature 409:363-66 (2001)). Further according to this non-limiting model, the siRNA duplexes are incorporated into a protein complex, followed by ATP-dependent unwinding of the siRNA, which then generates an active RNA-induced silencing complex (RISC) (WO 01/68836). The complex recognizes and cleaves a target RNA that is complementary to the guide strand of the siRNA, thus interfering with expression of a specific protein (Hutvagner et al., supra).
In C. elegans and Drosophila, RNAi may be mediated by long double-stranded RNA polynucleotides (WO 99/32619; WO 01/75164; Fire et al., 1998; Clemens et al., Proc. Natl. Acad. Sci. USA 97:6499-6503 (2000); Kisielow et al., Biochem. J. 363:1-5 (2002); see also WO 01/92513 (RNAi-mediated silencing in yeast)). In mammalian cells, however, transfection with long dsRNA polynucleotides (i.e., greater than 30 base pairs) leads to activation of a non-specific sequence response that globally blocks the initiation of protein synthesis and causes mRNA degradation (Bass, Nature 411:428-29 (2001)). Transfection of human and other mammalian cells with double-stranded RNAs of about 18-27 nucleotide base pairs in length interferes in a sequence-specific manner with expression of particular polypeptides encoded by messenger RNAs (mRNA) containing corresponding nucleotide sequences (WO 01/75164; Elbashir et al., 2001; Elbashir et al., Genes Dev. 15:188-200 (2001)); Harborth et al., J. Cell Sci. 114:4557-65 (2001); Carthew et al., Curr. Opin. Cell Biol. 13:244-48 (2001); Mailand et al., Nature Cell Biol. Advance Online Publication (Mar. 18, 2002); Mailand et al. 2002 Nature Cell Biol. 4:317).
siRNA polynucleotides may offer certain advantages over other polynucleotides known to the art for use in sequence-specific alteration or modulation of gene expression to yield altered levels of an encoded polypeptide product. These advantages include lower effective siRNA polynucleotide concentrations, enhanced siRNA polynucleotide stability, and shorter siRNA polynucleotide oligonucleotide lengths relative to such other polynucleotides (e.g., antisense, ribozyme or triplex polynucleotides). By way of a brief background, “antisense” polynucleotides bind in a sequence-specific manner to target nucleic acids, such as mRNA or DNA, to prevent transcription of DNA or translation of the mRNA (see, e.g., U.S. Pat. No. 5,168,053; U.S. Pat. No. 5,190,931; U.S. Pat. No. 5,135,917; U.S. Pat. No. 5,087,617; see also, e.g., Clusel et al., 1993 Nuc. Acids Res. 21:3405-11, describing “dumbbell” antisense oligonucleotides). “Ribozyme” polynucleotides can be targeted to any RNA transcript and are capable of catalytically cleaving such transcripts, thus impairing translation of mRNA (see, e.g., U.S. Pat. No. 5,272,262; U.S. Pat. No. 5,144,019; and U.S. Pat. Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246; U.S. 2002/193579). “Triplex” DNA molecules refers to single DNA strands that bind duplex DNA to form a colinear triplex molecule, thereby preventing transcription (see, e.g., U.S. Pat. No. 5,176,996, describing methods for making synthetic oligonucleotides that bind to target sites on duplex DNA). Such triple-stranded structures are unstable and form only transiently under physiological conditions. Because single-stranded polynucleotides do not readily diffuse into cells and are therefore susceptible to nuclease digestion, development of single-stranded DNA for antisense or triplex technologies often requires chemically modified nucleotides to improve stability and absorption by cells. siRNAs, by contrast, are readily taken up by intact cells, are effective at interfering with the expression of specific polypeptides at concentrations that are several orders of magnitude lower than those required for either antisense or ribozyme polynucleotides, and do not require the use of chemically modified nucleotides.
Importantly, despite a number of attempts to devise selection criteria for identifying oligonucleotide sequences that will be effective in siRNA based on features of the desired target mRNA sequence (e.g., percent GC content, position from the translation start codon, or sequence similarities based on an in silico sequence database search for homologues of the proposed siRNA) it is presently not possible to predict with any degree of confidence which of myriad possible candidate siRNA sequences that can be generated as nucleotide sequences that correspond to a desired target mRNA (e.g., dsRNA of about 18-27 nucleotide base pairs) will in fact exhibit siRNA activity (i.e., interference with expression of the polypeptide encoded by the mRNA). Instead, individual specific candidate siRNA polynucleotide or oligonucleotide sequences must be generated and tested to determine whether interference with expression of a desired polypeptide target can be effected. Accordingly, no routine method exists in the art for designing a siRNA polynucleotide that is, with certainty, capable of specifically altering the expression of a given PTP polypeptide, and thus for the overwhelming majority of PTPs no effective siRNA polynucleotide sequences are presently known.
Currently, therefore, desirable goals for therapeutic regulation of biological signal transduction include modulation of PTP (e.g., PTP-1B, DSP-3, SHP-2, KAP, PRL-3, cdc14 or cdc25 or other PTP)-mediated cellular events include, inter alia, inhibition or potentiation of interactions among PTP-binding molecules, substrates and binding partners, or of other agents that regulate PTP activities. Accordingly, a need exists in the art for an improved ability to intervene in the regulation of phosphotyrosine signaling, including regulating PTPs by altering PTP catalytic activity, PTP binding to PTP substrate molecules, and/or PTP-encoding gene expression. An increased ability to so regulate PTPs may facilitate the development of methods for modulating the activity of proteins involved in phosphotyrosine signaling pathways and for treating conditions associated with such pathways. The present invention fulfills these needs and further provides other related advantages.